MicroRNAs (miRNAs) are small non-coding RNAs whose precursors are transcribed from endogenous miRNA genes. miRNA precursors are processed by drosha in the nucleus and by dicer in the cytoplasm to generate mature double-stranded 19-23 bp miRNAs that are then loaded onto Argonaute-containing RNA-induced silencing complexes (RISC's), which in turn target the 3' UTRs of target mRNAs via imperfect base- pairing with these mRNAs, and thereby primarily inhibit translation of these mRNAs. Some miRNAs may target 100 or more mRNAs. Emerging evidence indicates that miRNAs may be key regulators of gene expression during normal development and under pathological conditions. However, it has been a major problem to identify the mRNA targets of a particular miRNA. Here, we propose a novel approach to identify mRNA targets for individual miRNAs in neurons. By doing so, this approach will significantly advance our understanding of the mechanisms by which miRNAs regulate neuronal development, synaptic function, and plasticity. Our novel approach proposes to co-immunoprecipitate a miRNA with its target mRNAs within the RISC complex, and to identify the co-immunoprecipitated targets by array and sequencing techniques. The main challenge here is how to immunoprecipitate only a given miRNA- RNP effector complex. To solve this problem, we propose to use conditional Dicer knockout mice (Dicer-/-), cultured neurons from these mice, and delete dicer in these neurons using lentivirally administered cre-recombinase, a standard technique in our laboratory. Thus we will have neurons that express no processed miRNAs, allowing us to reintroduce a given miRNA in mature form by transfection. In such neurons, the only miRNA-RNP complexes that are formed are those of our particular miRNA, making it possible for us to precisely define the mRNA targets for this particular miRNA. Using this method, one by one, we will be able to document mRNA targets of all of the individual microRNAs expressed in cultured cortical neurons. Moreover, this novel approach can easily be adapted to other types of neurons (e.g., striatal, cerebellum, hippocampal, or spinal motor neurons) or other systems and will significantly push both the miRNA and neuroscience research fields forward. Statement MicroRNAs are important regulators of gene expression that are encoded by our genome, and play a critical role in development and in the pathogenesis of several diseases. Individual microRNAs target specific sets of messenger RNAs to inhibit protein synthesis. However, it is extremely difficult to identify microRNA targets. Here we propose a novel approach to tackle this issue. If successful, this approach will revolutionize our view on the diverse biological functions of microRNAs. [unreadable] [unreadable]